Title Page
Abstract
Contents
1. Introduction 20
1.1. Nanoparticle-based tumor targeted drug delivery system 20
1.2. Limitations of EPR-based tumor targeted nanoparticles 21
1.3. Renal clearable nanocarriers for drug delivery 24
1.4. Cyclodextrin-based nanocarriers for tumor targeted delivery 25
1.5. Renal clearable cyclodextrin nanoplatforms for anticancer drug delivery to colorectal cancer 28
2. Materials and Methods 30
2.1. Materials 30
2.2. Synthesis and characterization of CD derivatives 31
2.3. Biodistribution studies of CD derivatives 42
2.4. Fabrication and application of CD/drug inclusion complexes 47
2.5. Statistical analysis 54
3. Results and Discussion 55
3.1. Design, synthesis, and optimization of CD derivatives 55
3.2. Biodistribution of CD derivatives 58
3.3. Tumor targetability, pharmacokinetics, and urinary excretion of PBA-(ZW)-CD/ACy7 60
3.4. Preparation and characterization of PBA-(ZW)-CD/drug inclusion complexes 63
3.5. Biodistribution of PBA-(ZW)-CD/drug inclusion complexes 66
3.6. PBA-(ZW)-CD/drug-assisted single-drug and combination therapies in HT-29 tumor xenograft mouse model 69
3.7. PBA-(ZW)-CD/drug combination therapy in orthotopic CRC mouse model 71
4. Conclusions 74
5. References 75
국문초록 145
Table 1. Physicochemical properties of five candidates with high colloidal stability. 96
Table 2. Pharmacokinetic parameters of free ACy7 and PBA-(ZW)-CD/ACy7 after intravenous injection at an ACy7 dose of 1 μmol kg¯¹. Data... 97
Table 3. Physicochemical properties, including number-weighted size distribution, mean hydrodynamic diameter (HD), and zeta potential, of... 98
Table 4. Complete blood counts of male BALB/c mice (5 weeks old) after intravenous administration of excess PBA-(ZW)-CD (270 mg kg¯¹ day¯¹,... 99
Table 5. In vitro antitumor efficacies of free drug and PBA-(ZW)-CD/drug inclusion complexes in HT-29 cells. Half-maximal inhibitory concentration... 100
Table 6. Blood biochemistry parameters after five rounds of treatment (3 mg kg¯¹ as each drug) (refer to Figure 34A). Data are presented as the mean ± SD... 101
Figure 1. Synthesis of sulfobetaine 2 (A₁) and 4 (A₂) that have one and two methylene groups between quaternary ammonium and carboxylic acid,... 103
Figure 2. Schematic view of CD derivatization. The carboxylic acid of charged moieties (Am, B, C, and D) was linked to the amine group of...[이미지참조] 104
Figure 3. Structures of cyclodextrin derivatives (CD 1-20). 105
Figure 4. Synthetic schemes of cyclodextrin derivatives (CD 1-20). (A) Preparation of PBA moieties (compounds 6 and 10). (B) Synthesis of PBA-... 108
Figure 5. ¹H-NMR (A) and ESI-MS (B) spectra of compounds 6 and 10. Samples were dissolved in DMSO-d₆/D₂O for ¹H-NMR analysis. 110
Figure 6. ¹H-NMR spectra of CD intermediates without spacers in the P2 position (compounds 11 and 12). Samples were dissolved in DMSO-d₆ for... 111
Figure 7. ¹H-NMR spectra of CD intermediates with PEG₄ (A; compounds 13, 15, 17, and 19) or PEG₈ (B; compounds 14, 16, 18, and 20) spacer in the... 113
Figure 8. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)spectra of CD intermediates conjugated with PEG (compounds 15 and... 114
Figure 9. ¹H-NMR spectra of CD intermediates with the caproic acid-PEG spacer in the P2 position (compounds 27, 28, 29, and 30). Samples were... 115
Figure 10. (A) Degree of PBA moiety conjugation was determined based on alizarin red S (ARS)-PBA moiety interaction. (B) Upon increasing PBA... 116
Figure 11. Percentage of charged moiety conjugation was determined by quantifying the residual amines via fluorescamine assay. (A) Fluorescence... 117
Figure 12. ¹H-NMR spectra of CD16-20. Samples were dissolved in DMSO-d₆/D₂O (9:1, v/v). Signal of Glu α-position proton appeared at 4.1-4.2 ppm. 118
Figure 13. Solubility screening of CD derivatives via turbidimetric analysis. Normalized absorbance values at 600 nm (OD₆₀₀ nm) of CD derivatives were... 119
Figure 14. Relationship between CD structure and colloidal stability. Turbidimetry-based solubility test was performed on CD derivatives... 120
Figure 15. (A) Synthetic schemes and ESI-MS spectra of adamantyl sulfocyanine (ACy)-7 and ACy5. (B) Visual appearance of free ACy7, CD... 121
Figure 16. Biodistribution of CD 16-20/adamantyl sulfocyanine 7 (ACy7) inclusion complexes. Ex vivo near-infrared fluorescence (NIRF) images of... 122
Figure 17. Tumor-to-background ratios of CD 16-20/ACy7 in each organ. Radiant efficiency values in tumor tissues are presented in the inset. 123
Figure 18. (A) Addition of the PEG₄ spacer in the P3 position significantly improved the tumor accumulation of zwitterionic CD derivatives, presumably... 124
Figure 19. (A) Structural components of PBA-(ZW)-CD/ACy7, including hydrophobic linker (caproic acid), PEG spacers, zwitterionic moiety... 125
Figure 20. Cellular uptake of PBA-(ZW)-CD inclusion complexes. (A) Fluorescence images of HT-29 cells after 2 h incubation with ACy7 or PBA-... 126
Figure 21. Tumor targetability and urinary excretion of PBA-(ZW)-CD/ACy7. (A) In vivo real-time NIRF imaging after intravenous... 127
Figure 22. Real-time tracing of PBA-(ZW)-CD after intravenous injection. SCy7-NHS (1 equiv.) was conjugated to one arm of compound 30, and... 128
Figure 23. ACy7 blood concentration versus time profiles of free ACy7 and PBA-(ZW)-CD/ACy7 after intravenous administration at an ACy7 dose of 1... 130
Figure 24. Schematic illustration of synergistic antitumor efficacy by DOX and UXT combination. UXT, a potent ERK inhibitor, may counteract the... 131
Figure 25. Preparation of PBA-(ZW)-CD/DOX and PBA-(ZW)-CD/UXT inclusion complexes via lyophilization. 132
Figure 26. (A) Proton nuclear magnetic resonance (¹H-NMR) spectra of free drugs and inclusion complexes. Slight changes in the chemical shifts of DOX... 133
Figure 27. (A) Number-weighted size distribution of inclusion complexes at pH 5.5 and 7.4. (B) Drug release profiles of PBA-(ZW)-CD/DOX and PBA-... 134
Figure 28. Biodistribution of PBA-(ZW)-CD/drug inclusion complexes. (A) DOX distribution in normal tissues related to drug adverse effects and tumor... 135
Figure 29. Intratumoral distribution of PBA-(ZW)-CD/drug inclusion complexes. (A) Matrix assisted laser desorption/ionization-mass... 136
Figure 30. In vitro toxicity studies of blank PBA-(ZW)-CD. HT-29 cell viability after 24, 48, and 72 h incubation with PBA-(ZW)-CD at various... 137
Figure 31. PBA-(ZW)-CD/doxorubicin (DOX)-assisted single-drug therapy in heterotopic HT-29 xenograft mice (n = 8). (A) Tumor growth profiles over... 138
Figure 32. PBA-(ZW)-CD/UXT-assisted single-drug therapy in heterotopic HT-29 xenograft mice (n = 5). (A) Tumor growth profiles over 14 days. PBS,... 139
Figure 33. Cell viability profiles of free drug and PBA-(ZW)-CD/drug inclusion complexes in HT-29 cells at 48 h incubation. Data are presented as... 140
Figure 34. PBA-(ZW)-CD/drug-assisted combination therapy in colorectal cancer (CRC) models. (A) Tumor growth profile of heterotopic HT-29... 141
Figure 35. Body weight changes of heterotopic HT-29 xenograft mice during the experimental period (n = 8). 142
Figure 36. (A) In vivo bioluminescence images of orthotopic CRC model prepared via the inoculation of HT-29/Luc cells into BALB/c nude mice (n =... 143
Figure 37. Hematoxylin and eosin (tumor and organs) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL; tumor)... 144